Device and method for quantitatively determining an analyte, a method for determining an effective size of a molecule, a method for attaching molecules to a substrate, and a device for detecting molecules

ABSTRACT

A device for quantitatively determining an analyte is provided to conspicuously improve the performance of the quantitative determination. This device is equipped with a flow channel, an analyte detecting unit for capturing and detecting the analyte, and a quantitative measurement unit for quantitatively determining the analyte, wherein a signal generated when the analyte detecting unit has detected the analyte is divided into a plurality of parts in the direction of the flow in the flow channel at the quantitative measurement unit for processing. Also provided are technologies including one for controlling the density of molecules attached to the surface of a solid. In these technologies, when molecules are attached to a substrate, the density of attached molecules is controlled, by having an electrolyte also present in a solution containing the molecules to adjust the screening effect by the electrolyte, and by taking into consideration the effective size of a molecule.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a Divisional application of prior application Ser. No.11/430,045, filed on May 9, 2006 now abandoned, which was a Divisionalof prior application Ser. No. 11/036,367, filed on Jan. 18, 2005, nowU.S. Pat. No. 7,169,617, which is hereby incorporated by reference.

This application is based upon and claims the benefit of priority fromthe prior Japanese Patent Application No. 2004-238980, filed on Aug. 19,2004 and No. 2004-283245, filed on Sep. 29, 2004, the entire contents ofwhich are incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method and device for quantitativelydetermining an analyte that is used for biochips, DNA chips, etc. aswell as a method for attaching molecules to a substrate that is used forbiochips, DNA chips, etc., and a device manufactured using the method.

2. Description of the Related Art

Recently nanotechnology has become a key word which has drawn muchattention from many people, caused by various factors including ananotechnology initiative proposed in United States in 2,000.Specifically, the nano-biotechnology field that is an integration ofsemiconductor microprocessing technologies (semiconductornanotechnologies) and biotechnologies is expected as a new technicalfield that may bring about drastic solutions to the conventionalproblems, and many researchers are working energetically in this field.

Among these, the biochip technologies represented by DNA chips (or DNAmicroarrays) attract attention as an effective means for gene analysis.Biochips comprise substrates made of glasses, silicon, plastics, etc. onthe surface of which numerous different test substances ofbiomacromolecules such as DNAs and proteins, are highly densely arrayedas spots. They can simplify examination of nucleic acids and proteins inthe fields of clinical diagnosis and pharmacotherapy (see, for example,Japanese Unexamined Patent Application Publication No. 2001-235468(paragraph numbers 0002-0009), and Annual Review of BiomedicalEngineering, vol. 4, p. 129-153, 2002, and Nature Biotechnology, vol.21, p. 1192-1199, 2003).

Devices, such as those chips, manufactured by integrating micromachiningtechnologies and sensing technologies under technologies for detecting atiny analyte, are generally called MEMSs or μTASs, which draw attentionas devices for greatly improving the detection sensitivity and detectiontime compared with the prior art. The term MEMS is the abbreviation ofMicro Electro Mechanical System, that is, a technology to preparemicroscopic matters, based on semiconductor processing technologies, ormicroscopic, precision devices prepared, using the technologies. Ingeneral, it is a system wherein a plurality of functional units such asmechanical, optical and hydrodynamic units are integrated andminiaturized. The term μ-TAS is an abbreviation of Micro Total AnalysisSystem, and is a chemical analysis system with micropumps, microvalves,sensors or the like, miniaturized, accumulated, and integrated.

The features of these devices are that it is possible to evaluate a verysmall amount of a sample containing an analyte, and that it is possibleto perform a real-time evaluation by making a sample flow into ananalyte detecting unit. There are many other advantages including onethat it is possible to evaluate a plurality of analytes at the sametime, by arranging analyte detecting units in parallel or in series.

Furthermore, regarding the technology that is a key for thesenano-biotechnologies, there is an issue how biomolecules such as DNAsand proteins should be attached to a solid such as a semiconductingmaterial and metal, in order to make the surface of the solid have aspecific function. Attaching of molecules to the surface of a solid byphysical adsorption represented by LB (Langmuir Brodgett) membranes havebeen long and widely known. However, the molecules formed only byphysical adsorption are stripped off as time passes, or by repetitiveuse. Accordingly, attachment of molecules by chemical adsorptionutilizing a chemical reaction between the surface of a solid andmolecules has been generally used recently. Specifically, a method forattaching molecules by chemical adsorption wherein an SH (thiol) groupis placed at an end of a molecule to utilize covalent bonding between S(sulfur) and a metal or semiconducting material, is proposed, and iswidely used in various researches and developments (see, for example,Chemical Reviews, vol. 96, p. 1533-1554, 1996).

When molecules having an alkyl chain which has an SH end group is usedas the molecules, a monomolecular film with a regular array of moleculescan be formed on a solid, by means of van der Waals force of the alkylchain. It is easy to form this membrane. That is, when the surface of asolid is immersed in a solution containing these molecules, amonomolecular film (self-assembled monolayer film) is spontaneouslyformed on the surface of the solid.

It is possible to form a monolayer film having a function on thesurface, by attaching DNAs, proteins, or functional groups having otherfunctions to a part of the alkyl chain (see, for example, BioconjugateChemistry, vol. 8, p. 31-37, 1997).

SUMMARY OF THE INVENTION

However, regarding the above-described device such as a MEMS and μTAS,though the presence or absence of an analyte can be detected when anextremely highly sensitive device is installed as the analyte detectingunit, there is a problem that the signal from the analyte detecting unitmay be saturated by a small amount of analyte, and accordingly it isdifficult to quantitatively determine the analyte, if the devicecharacteristics are not sufficiently linear to the amount of analyte (inother words, if the dynamic range is low for the amount of analyte).

Particularly in detecting biomolecules with a biochip or the like,molecules such as antibody molecules that are specifically adsorbed tothe biomolecules, are often used as the analyte detecting units. In sucha case, an analyte detecting unit and an analyte are often bound in a1:1 ratio, whereby a small number of analytes will saturate the analytedetecting units if the number of analyte detecting units is small,making it impossible to perform quantitative determination in a widerange. On the other hand, if the number of analyte detecting units isincreased to make it possible to perform quantitative determination in awide range, it will cause degradation of the detection sensitivity andincrease of background noises.

The first aspect of the present invention is directed to solving theabove-described problem, and providing a new technology with which it ispossible to quantitatively determine an analyte in a wide range withoutlowering the detection sensitivity, even when an analyte detecting unitis bound to an analyte in a 1:1 ratio, as is observed in the case of abiochip.

According to one embodiment of the first aspect of the presentinvention, provided is a device for quantitatively determining ananalyte equipped with a flow channel, an analyte detecting unit forcapturing and detecting the analyte, and a quantitative measurement unitfor quantitatively determining the analyte, wherein a signal generatedwhen the analyte detecting unit has detected the analyte is divided intoa plurality of parts in the direction of the flow in the flow channel atthe quantitative measurement unit for processing.

Using the device for quantitatively determining an analyte according tothis embodiment of the present invention makes it possible toquantitatively determine an analyte in a wide range without lowering thedetection sensitivity, with the result that the performance inquantitative determination of an analyte is conspicuously improved.

In this embodiment, preferable are that the analyte detecting unit hasan analyte capturing unit for capturing the analyte; that a plurality ofanalyte detecting units are disposed in the direction of the flow in theflow channel; that the length of the analyte detecting unit in thedirection of the flow in the flow channel is not less than twice thewidth of the flow channel; that a plurality of analyte detecting unitsare disposed in the direction along the width of the flow channel; thatthe quantitative measurement unit quantitatively determines the analyte,using an optical signal; that the quantitative measurement unitquantitatively determines the analyte, using an electric signal; thatthe thickness of the flow channel at the analyte detecting unit is notmore than 100 times the effective height of a captured analyte; that thethickness of the flow channel at the analyte detecting unit is madelarger as the flow goes downstream; that the analyte detecting unit isan electrode to which an electric potential can be applied forelectrically attracting charged analytes to the electrode; particularlythat the electric potential for electrically attracting charged analytesto the electrode can be changed according to the location of theelectrode in the direction of the flow; that a micropump,electrophoretic flow or electroosmotic flow is utilized to make asolution containing the analyte flow in the flow channel; that aplurality of analyte capturing units for specifically capturingdifferent analytes, are disposed on the analyte detecting unit; that theanalyte is a DNA, and the analyte capturing unit has a function to bespecifically bound to a DNA; and that the analyte is a protein, and theanalyte capturing unit has a function to be specifically bound to aprotein.

According to another embodiment of the first aspect of the presentinvention, provided is a method for quantitatively determining ananalyte comprising: using a flow channel, an analyte detecting unit forcapturing and detecting the analyte, and a quantitative measurement unitfor quantitatively determining the analyte; and dividing a signalgenerated when the analyte detecting unit has detected the analyte, intoa plurality of parts in the direction of the flow in the flow channel atthe quantitative measurement unit for processing.

Using the method for quantitatively determining an analyte according tothis embodiment of the present invention makes it possible toquantitatively determine an analyte in a wide range without lowering thedetection sensitivity, with the result that the performance inquantitative determination of an analyte is conspicuously improved.

In this embodiment, preferable are that the analyte detecting unit hasan analyte capturing unit for capturing the analyte; that a plurality ofanalyte detecting units are disposed in the direction of the flow in theflow channel; that the number of analytes captured by the analytedetecting unit in the direction of the flow in the flow channel isoptimized; more specifically that the optimization is performed bychanging at least one factor selected from the group consisting of thesupplying velocity of the analyte, the length of the analyte detectingunit in the direction of the flow in the flow channel, the number of theanalyte detecting units in the direction along the width of the flowchannel, and the thickness of the flow channel; that the quantitativemeasurement unit quantitatively determines the analyte, using an opticalsignal; that the quantitative measurement unit quantitatively determinesthe analyte, using an electric signal; that the analyte detecting unitis an electrode, and an electric potential for electrically attractingcharged analytes to the electrode, is applied to the electrode;particularly that the electric potential for electrically attractingcharged analytes to the electrode is changed according to the locationof the electrode in the direction of the flow; that a plurality ofanalyte capturing units for specifically capturing different analytes,are disposed on the analyte detecting unit; that the analyte is a DNA,and the analyte capturing unit has a function to be specifically boundto a DNA; and that the analyte is a protein, and the analyte capturingunit has a function to be specifically bound to a protein.

According to other embodiments of the first aspect of the presentinvention, provided are a MEMS and μTAS equipped with theabove-described device for quantitatively determining an analyte.

By the above-described first aspect of the present invention, it ispossible to quantitatively determine an analyte in a wide range withoutlowering the detection sensitivity, with the result that the performancein quantitative determination of an analyte is conspicuously improved.

Regarding the attachment of biomolecules, when the surface of a solid ismodified simply with molecules comprising an alkyl chain havingfunctional groups that have various functions, there is a problem thatit is difficult to control the density of the molecules comprising thealkyl chain attached to the surface (the density of molecules attachedto the surface of a solid such as a substrate is referred to as the“attaching density” in the present invention).

In addition, even though it is known that such chemical adsorption ofmolecules is dependent on diffusion, and there is an example reporting achange in the attaching density of molecules observed with time, a stateof a relatively low attaching density is attained in a short time, andtherefor, it is difficult to realize, with a high reproducibility, a lowattaching density with wide spacing between adjacent molecules for whichthe influence of interactions between molecules can be ignored, bycontrolling the time for preparation (see Nucleic Acids Research, vol.29, p. 5163-5168, 2001, for example).

Furthermore, biomolecules such as DNAs and proteins are relatively largecompared with usual molecules having an alkyl chain. Accordingly, if aself-assembled monolayer film that is compact (that is, having a highdensity of biomolecules) is simply prepared, steric hindrance betweenbiomolecules becomes large, posing a problem of hindering free motion ofthe biomolecules. On such a surface, it is not possible to sufficientlyacquire signals related to reactions between the biomolecules, theirthermal motions (signals related to fluctuating motion andbending/stretching motions, for example), etc.

On the other hand, in the field of biosensors which utilize reactionsbetween such molecules and/or motions of molecules for a sensingtechnology, it is very important to control the attaching density and/orto realize a low attaching density with a high reproducibility.Accordingly, there is a strong need for a technology to control thedensity of molecules attached to the surface of a solid.

A DNA has a negative charge in an aqueous solution on the phosphoricacid group on its back bone. Accordingly, DNA moleculeselectrostatically repel each other in an aqueous solution. In an aqueoussolution containing an electrolyte, it is also known that ions having apositive charge surround a DNA molecule, indicating an action ofcompensating the electric charge of the DNA molecule (called thescreening effect, or Debye effect).

This screening effect is dependent on the concentration of anelectrolyte. That is, it is possible to control the closest distancebetween DNA molecules, by controlling the concentration of anelectrolyte, thus providing a possibility of controlling the eventualattaching density of molecules having an electric charge such as DNAmolecules.

However, though there are some reports confirming such an effect, theyare only qualitative studies, and cannot predict the attaching density(see Journal of American Chemical Society, vol. 119, p. 8916-8920, 1997,for example).

Furthermore, much is unknown what structure molecules in the shape of astrand such as DNS molecules have in an aqueous solution, which makes itall the more difficult to handle such molecules.

The second aspect of the present invention is directed to solving theabove-described problems, and to provide a technology to control thedensity of molecules attached to the surface of a solid.

It is also directed to providing technologies that can clarify thestructure and behavior of molecules attached to the surface of a solidin a solution, and realize a device using molecules attached to thesurface of a solid, with high reliability and high sensitivity.

Other objects and advantages of the present invention will be clarifiedin the following explanation.

According to one embodiment of the second aspect of the presentinvention, provided is a method for determining an effective size of amolecule having an electric charge, wherein the effective size of amolecule having an electric charge in a solution containing anelectrolyte and the molecule having an electric charge is estimated,using the screening effect by the electrolyte.

By this embodiment of the present invention, it is possible to know theeffective size of a molecule having an electric charge for controllingthe attaching density of molecules attached to the surface of a solid.

According to another embodiment of the second aspect of the presentinvention, provided is a method for attaching molecules having anelectric charge to a substrate, wherein the attaching density ofmolecules is controlled, by having an electrolyte present in a solutioncontaining the molecules to adjust the screening effect by theelectrolyte, and by taking into consideration the effective size of amolecule in a solution, when the molecules having an electric charge areattached to the substrate.

By this embodiment of the present invention, it is possible to realize arequired attaching density.

It is preferable that the effective size of a molecule having anelectric charge in a solution containing an electrolyte and the moleculehaving an electric charge is estimated from the screening effect by theelectrolyte as explained above, and the thus obtained effective size isused as the above-described effective size.

In addition, while any electrolyte may be used in any of theabove-described embodiments, preferable are that an electrolyte composedof a monovalent cation and a monovalent anion is used as theelectrolyte; and that the electrolyte comprising a monovalent cation anda monovalent anion is NaCl, KCl or a mixture thereof.

According to still another embodiment of the second aspect of thepresent invention, provided is a device for attaching molecules havingan electric charge to a substrate, wherein the density of moleculesattached to the substrate can be controlled, using the method forattaching molecules to a substrate according to the above-describedmethod. By this embodiment of the present invention, it is possible toobtain a substrate having a desired attaching density.

According to still another embodiment of the second aspect of thepresent invention, provided is a device for detecting molecules, whereinthe above-described method for attaching molecules to a substrate isused, and a substrate of which the attaching density is controlled sothat the distance between adjacent molecules attached to the substrateis not less than twice or less than twice the effective length of amolecule, is used as a molecule detecting unit.

By this embodiment of the present invention, it is possible to clarifythe structures and behaviors of molecules attached to the surface of asolid in a solution, and realize a device with high reliability and highsensitivity.

It is preferable that the substrate is made of an electroconductivematerial, a semiconducting material, or an insulating material, andparticularly made of gold or platinum.

Also preferable in each embodiment of the present invention are that themolecule having an electric charge comprises a material selected fromthe group consisting of proteins, DNAs, RNAs, antibodies, natural orartificial single-stranded nucleotides, natural or artificialdouble-stranded nucleotides, aptamers, products obtained by limiteddecomposition of antibodies with a protease, organic compounds havingaffinity to proteins, biomacromolecules having affinity to proteins,complex materials thereof, ionic polymers charged positively ornegatively, and arbitrary combinations thereof; that the molecule havingan electric charge comprises a thiol group; and that the molecule havingan electric charge comprises a fluorescent pigment.

By this embodiment of the present invention, it is possible to controlthe attaching density of molecules attached to the surface of a solid,whereby it is possible to clarify the structure and behavior of themolecules attached to the surface of a solid in a solution. It is alsopossible to realize a device using the molecules attached to the surfaceof a solid with high reliability and high sensitivity.

Each of the above-described two aspects of the present invention can beapplied to the other aspect. For example, the device according to thefirst aspect can be used for determining an effective size of a moleculehaving an electric charge, and the method for attaching molecules havingan electric charge to a substrate according to the second aspect can beused for a detecting unit in the device according to the first aspect.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1-A is a schematic view explaining how an analyte is detected by aconventional device for quantitatively determining an analyte;

FIG. 1-B is a schematic view explaining how an analyte is detected by aconventional device for quantitatively determining an analyte;

FIG. 1-C is a schematic view explaining how an analyte is detected by aconventional device for quantitatively determining an analyte;

FIG. 2-A is a schematic view explaining how an analyte is detected by aconventional device for quantitatively determining an analyte;

FIG. 2-B is a schematic view explaining how an analyte is detected by aconventional device for quantitatively determining an analyte;

FIG. 2-C is a schematic view explaining how an analyte is detected by aconventional device for quantitatively determining an analyte;

FIG. 3-A is a schematic view explaining how an analyte is detected by adevice for quantitatively determining an analyte according to thepresent invention;

FIG. 3-B is a schematic view explaining how an analyte is detected by adevice for quantitatively determining an analyte according to thepresent invention;

FIG. 3-C is a schematic view explaining how an analyte is detected by adevice for quantitatively determining an analyte according to thepresent invention;

FIG. 4-A is a schematic view explaining how an analyte is detected by adevice for quantitatively determining an analyte according to thepresent invention;

FIG. 4-B is a schematic view explaining how an analyte is detected by adevice for quantitatively determining an analyte according to thepresent invention;

FIG. 4-C is a schematic view explaining how an analyte is detected by adevice for quantitatively determining an analyte according to thepresent invention;

FIG. 5 is a schematic view explaining the thickness of a flow channeland the height of an analyte;

FIG. 6 is a schematic view explaining a device for quantitativelydetermining an analyte for which no capturing unit is installed.

FIG. 7 is a graph showing the dependency of a Debye length on the ionconcentration of the electrolyte, when a 1:1 type analyte is used;

FIG. 8 is a schematic view showing the closest packing structure ofmolecules attached to a substrate, and the principle to calculate theattaching density;

FIG. 9 is a schematic view showing the closest packing structure ofmolecules attached to a substrate, and the principle to calculate theattaching density, when the Debye length is taken into consideration;

FIG. 10 is a graph showing the dependency of the attaching density ofadsorbed molecules on the ion concentration of the electrolyte, when theattaching density is calculated, taking the Debye length intoconsideration;

FIG. 11 is a graph showing the relationship between the attachingdensity of single-stranded oligonucleotide molecules and the ionconcentration of the electrolyte, and the relationship between the Debyelength and the ion concentration of the electrolyte;

FIG. 12 is a graph showing the relationship between the fluorescenceintensity and the attaching density; and

FIG. 13 is a schematic view showing an example of a device for detectingmolecules, wherein a fluorescent pigment is attached to chargedmolecules for the fluorescence to be detected.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Embodiments according to the present invention will now be describedusing drawings, examples, etc. These drawings, examples, etc., anddescriptions are for demonstrating the present invention, and do notlimit the scope of the invention. Needless to say, other embodiments canbe included in the scope of the present invention as long as theyconform to the essential character according to the present invention.The same reference numeral indicates the same element in the drawings.

The device for quantitatively determining an analyte according to thefirst aspect of the present invention, is equipped with a flow channel,an analyte detecting unit (also referred to as a “detecting unit”hereinafter) for capturing and detecting the analyte, and a quantitativemeasurement unit for quantitatively determining an analyte, wherein asignal generated when the detecting unit has detected an analyte isdivided into a plurality of parts in the direction of the flow in theflow channel at the quantitative measurement unit for processing. Bythis, it is possible to quantitatively determine an analyte with a highsensitivity and a high dynamic range.

Any signal may be used as a signal that is processed at the quantitativemeasurement unit for quantitatively determining the analyte. However, anelectric signal or optical signal is highly reliable, and accordinglypreferable.

Any method may be used as a method for the detecting unit to capture theanalyte. A method in which the detecting unit has an analyte capturingunit for capturing an analyte (also simply referred to as “capturingunit”, hereinafter) is the easiest method, and is preferable. Althoughthe detecting unit and the capturing unit are generally separatelyexplained in the following explanation, it is permissible to include thecapturing unit into the detecting unit and call the whole of them as adetecting unit, as long as it does not contradict the gist of thepresent invention.

Any method may be used as a method in which the quantitative measurementunit can divide the signal generated when the detecting unit hasdetected the analyte, into plural parts for processing in the directionof the flow in the flow channel. When the quantitative measurement unitquantitatively determines an analyte using an optical signal, an opticalsignal such as fluorescence over the detecting unit may be scanned, oran optical signal over the detecting unit may be received as a whole,and the image is divided into plural parts for analysis.

In the case of an optical signal, it is easy to observe it by dividingit into plural parts, without dividing the source of the signal intoplural parts beforehand. However, when the quantitative measurement unitquantitatively determines an analyte by an electric signal, it isnecessary to install a plurality of separate detecting units in thedirection of the flow in the flow channel so as to take out a pluralityof electric signals.

The term “in the direction of the flow in the flow channel” refers to“in the direction along which a medium containing an analyte flows inthe flow channel”, and the act of “dividing into plural parts in thedirection of the flow in the flow channel for processing” may be, forexample, realized by scanning an optical signal in the direction of theflow in the flow channel, or by installing a plurality of detectingunits in the direction of the flow in the flow channel, and taking outelectric signals from each of the detecting units. It is to be notedthat the “direction of the flow in the flow channel” in this case is notnecessarily a straight line direction. For example, it is not necessaryto install a plurality of detecting units on a straight line. Suchsignals may be combined with scanning of an optical signal in thedirection along the width of the flow channel, and when there are aplurality of detecting units in the direction along the width of theflow channel, electric signals from these detecting units may be usedtogether with the above-described signals.

The following is an explanation of a case in which a capturing unit isinstalled on a detecting unit, and a quantitative measurement unitquantitatively determines an analyte by an electric signal, in referenceto FIGS. 1-3.

FIGS. 1-3 are schematic views of devices for quantitatively determiningan analyte for explaining the principles of conventional examples andthe first aspect of the present invention. In FIGS. 1-3, a device forquantitatively determining an analyte 11 for uses such as MEMSs andμTASs has a detecting unit 13 equipped with a capturing unit 14 thatspecifically binds to an analyte in a 1:1 ratio in a flow channel 12,and generates an electric signal when bound.

A conventional device for quantitatively determining an analyte will beexplained, using FIGS. 1-A to 1-C, and FIGS. 2-A to 2-C. The device forquantitatively determining an analyte 11 is equipped with one capturingunit on the detecting unit as shown in FIG. 1-A, a solution forinspection (inspection solution) 18 comprising an analyte 19 flows inthe flow channel as shown in FIG. 1-B, and when it passes through aninspection unit 13, the analyte 19 is specifically bound to thecapturing unit 14, generating an electric signal, as shown in FIG. 1-C.This signal is taken out through a signal receiving electrode 15,necessary processing (amplification, conversion, etc.) is performed at asignal processing circuit 16, and ascertained as a changing signal on amonitor 17.

When one capturing unit 14 is used for the detecting unit 13, as is thecase for the conventional examples, only one of the analytes is bound tothe capturing unit, generating a signal as shown in FIG. 2-B, when asolution 18 containing two or more analytes 19 flows into the flowchannel 12 as shown in FIG. 2-A. Accordingly, the remaining analytesflow out of the flow channel, without being bound to the capturing unit,with the result that it is impossible to quantitatively determine theanalytes precisely.

Furthermore, when three capturing units are installed on the detectingunit to increase the capacity of quantitative determination three times,for example, as shown in FIG. 2-C, it is necessary to raise the dynamicrange to correspond to the electric signal that is three times as large(a monitor 17 shown at the upper side of the figure), or decrease thesensitivity to one third (a monitor 17 shown at the lower side of thefigure).

To compare, the “detecting unit” is divided into plural parts in thedirection of the flow in the flow channel for the processing, accordingto the present invention. For example, FIG. 3-A shows five detectingunits 13 each having one capturing unit 14 (designated by numerals 1-5)that are electrically separated from each other, are installed in seriesin the direction of the flow in the flow channel.

In addition, a switching electrode 31 is installed to monitor signalsfrom the electrodes in a alternate manner. In this way, when analytesflown in the flow channel are bound to the capturing units sequentiallyfrom the upstream side to the downstream side as shown in FIG. 3-B,signals are generated sequentially from the upper detecting unit to thelower detecting unit according to the amount (that is number) of theanalytes.

In such a case, the signals from the detecting units represented bycircled numbers 1-5 in the figure obtained by the sequential switchingas shown in FIG. 3-C are subjected to necessary processing(amplification, conversion, etc.) by the signal processing circuit 16.When the amount (or number) of the analytes in the solution is three,signals from the uppermost detecting unit to the third one are detectedthrough continuous monitoring by the monitor 17, thus making it possibleto quantitatively determine the amount of analytes by the locations ofthe detecting units through which signals are observed. These switchingelectrode 31, signal processing circuit 16 and monitor 17 constitute thequantitative measurement unit according to the present invention.

Utilizing the present invention, it is possible to increase the capacityfor quantitatively determining an analyte by combining a conventionalsignal processing circuit with a simple switch, for example, and improvethe dynamic range of the detecting device without lowering thesensitivity.

The above explanation is made to a case when one capturing unit isinstalled on a detecting unit. Similar effect can also be provided whena plurality of capturing units are installed on a detecting unit, takinginto consideration a case when a large number of analytes are present.Generally speaking, as it is not easy to install only one capturing uniton a detecting unit, it is often practical to install a plurality ofcapturing units on a detecting unit. In this case, because the detectionsensitivity decreases when too many numbers of capturing units areinstalled on a detecting unit, it is often preferable to make the numberof the capturing units installed on a detecting unit as small aspossible, by making the size of the detecting unit smaller, making thedensity of capturing units smaller, or employing other similar measures.

When analytes are captured directly by a detecting unit without anintervening capturing unit, a case may be acceptable in which onedetecting unit can capture a plurality of analytes similarly to theabove. However, it is also often preferable to make the number of theanalytes that can be captured by one detecting unit as small aspossible, by making the size of the detecting unit smaller, or employingother similar measures.

When optical signals are utilized, each of analytes captured by acapturing unit emits an optical signal. Accordingly, it is not necessaryto install plural detecting units, and it is sufficient if thequantitative measurement unit can divide these signals into a pluralityof parts in the direction of the flow in the flow channel forprocessing.

FIGS. 4-A to 4-C are schematic views of devices for quantitativelydetermining an analyte where analytes captured by capturing units emitfluorescence. In FIGS. 4-A to 4-C, capturing units that have capturedanalytes and capturing units that have not captured an analyte areshown. In this case, there are a plurality of capturing units both inthe direction of the flow and along the width of the flow channel in onedetecting unit. In such a case, the quantitative measurement unit canalso divide the signals obtained by the scanning in the direction of theflow in the flow channel, into plural signals in the direction along thewidth of the flow channel for processing. When there happen to be a casewhen the numbers of capturing units that have captured analytes in thedirection of the flow in the flow channel, are different in thedirection along the width of the flow channel, as shown in FIG. 4-C, itis reasonable to use the average value for the quantitativedetermination, for example.

It is not always necessary for the quantitative measurement unit todivide optical signals for processing according to each analyte, eachcapturing unit, or each detecting unit. For example, it may be possibleto put some analytes, capturing units, and/or detecting units togetherinto a group in the direction of the flow to be subjected to theprocessing. For example, signals in a certain wavelength range may begrouped for the processing.

It is not always necessary to install a capturing unit if a detectingunit can directly capture an analyte. A case where an analyte isphysically or electrically adsorbed onto a detecting unit is such acase. However, since such adsorption is generally weak, it is preferableto install a capturing unit that can capture an analyte on the detectingunit. When a plurality of capturing units are used for specificallycapturing different analytes, different types of plural analytes can bequantitatively determined at the same time. Accordingly, it ispreferable.

It is preferable that the length of the detecting unit or detectingunits in the direction of the flow is not less than twice the width ofthe flow channel. If there is one detecting unit in the direction of theflow, the length of the detecting unit in the direction of the flow isits length, and if there are plural detecting units, the length is thetotal length. By this, it is easier to capture analytes with a pluralityof capturing units or detecting units in the direction of the flow, thusincreasing the detection sensitivity.

When the width of the flow channel is larger than the width of onedetecting unit, a plurality of detecting units may be installed in thedirection along the width of the flow channel. Since the flow channelaccording to the present invention is as tiny as is seen from the factthat the width is in a range of from 100 μm to 5 mm and the height is ina range of from 1 μm to 1 mm, for example, and the flow rate isgenerally as small as not more than 10 cm/second, which is in the regionof laminar flow, it is not always necessary to install a plurality ofdetecting units in the direction along the width of the flow channel.However, it is preferable to realize quantitative determination with ahigher precision. For example, by subjecting signals obtained byscanning in the longitudinal direction of the flow channel, to averagingin the direction along the width, the same number of data can beobtained through one measurement as those obtained by repeating pluraltimes of measurement with a device for quantitatively determining ananalyte that has only one detecting unit in the direction along thewidth of the flow channel.

While the above explanation is made on cases where analytes are bound tocapturing units sequentially from the upstream side to the downstreamside of the flow, it is not an essential requirement. Depending on thecapturing units and analytes for use, there may be cases wherein theease and velocity of bonding are not sufficient for the flow rate of theanalytes, and therefore, the analytes may not be captured sequentiallyfrom the capturing unit in the upstream side to the capturing unit inthe downstream side of the flow. In such a case, if the number or thelength of detecting units installed as are divided into plural parts inthe direction of the flow in the flow channel is large enough, precisedetermination is possible as long as all the analytes are bound to thecapturing units, though they may not be bound to the capturing unitssequentially from the upstream side to the downstream side.

In addition, it is also effective to make the thickness of the flowchannel at the detecting unit sufficiently thin compared with theeffective height of analytes, and/or make the flow rate smallersequentially by making the thickness of the flow channel thicker as theflow goes down, thus securing the time for analytes that become smallerin number to be bound to capturing units, while the number of detectingunits arrayed in series in the direction of the flow of the flow channelis not increased, or the number is increased.

It was found preferable that the thickness of the flow channel is madeto be not more than 100 times the effective height of a capturedanalyte, in order to fully capture the analyte. The effective height ofan analyte is the height from the surface of the detecting unit to thehighest part of the analyte. The height of the capturing unit isincluded in the definition, as shown in FIG. 5. FIG. 5 is a schematicpartial cross-sectional view of a device for quantitatively determiningan analyte, for explaining the height 51 of an analyte 19, taking intoconsideration the height of a capturing unit 14. The “thickness of theflow channel” 52 in this case is the height from the surface of thedetecting unit 53 to the ceiling part of the flow channel 54. If theheight of the surface of the detecting unit is located higher than thebottom surface 55 of the flow channel, it is preferable that thedetecting unit is installed to the full width of the flow channel, orthe sections of the flow channel that are adjacent to the sides of thedetecting unit (in the direction along the width of the flow channel)and on which the detecting unit is not located, have a raised bottomsurface that is as high as the surface of the detecting unit or higher.

The term “effective” refers to the average value of height when ananalyte is actually captured by a detecting unit (numeral 51 in FIG. 5).When the analyte and the capturing unit are molecules, the shapes aretaken into consideration. When the analyte and the capturing unit aremolecules, it is generally difficult to know the effective height. Insuch a case, the effective height of an analyte may be determined byemploying, as the effective length of the molecule, a value that is50-80% of the largest length which is obtained when the molecule isstretched.

Furthermore, it is preferable that the thickness of flow channel at thedetecting unit is made thicker as the flow goes downstream. There may bea case in which depending on capturing units and analytes for use, theease and velocity of bonding are not sufficient for the flow rate, andtherefore, analytes cannot be bound to the capturing units sequentiallyfrom the upstream side to the downstream side of the flow, in a thickflow channel, with the result that the quantitative determination ishindered. The above-described flow channel is particularly useful insuch a case.

Furthermore, in a case where analytes are molecules which are wholly orpartly charged such as DNA and protein molecules, it is preferable thatthe detecting unit is an electrode, and an electric potential can beapplied to the electrode to electrically attract the charged analytes tothe electrode. In this way, binding of the analytes to the capturingunit can be accelerated by applying the electric potential to theelectrode, so as to shorten the detection time and improve the detectionsensitivity.

Furthermore, it is preferable to be able to change the electricpotential to electrically attract the charged analytes to the electrodeaccording to the location of the electrode in the direction of the flowin the flow channel. In this way, for example, it is possible toincrease the attracting effect by the electric potential as the flowgoes downstream and prevent the sensitivity from being reduced, bychanging the electric potential according to the location of theelectrode, since the concentration of the analytes in the medium becomessmaller as the flow goes downstream, reducing the analyte capturingefficiency.

In this way, it is possible to quantitatively determine an analyte oranalytes with a high sensitivity and a high dynamic range. When it isseen from the viewpoint of a method for quantitatively determining ananalyte, it can be considered important to optimize the number ofanalytes to be captured by the analyte detecting unit, in the directionof the flow in the flow channel. Hereupon, the term “to optimize” refersto selecting conditions for the quantitative determination so that thesensitivity and dynamic range in the quantitative determination ofanalytes are in a proper range corresponding to the actual purposes ofthe quantitative determination. Specifically, it is preferable to do theoptimization, by changing at least one factor selected from the groupconsisting of the supply rate of analytes, the length of the detectingunit in the direction of the flow in the flow channel, the number ofdetecting units in the direction along the width of the flow channel,and the thickness of the flow channel.

Of these, regarding the supply rate of analytes, if the fluctuation ofthe flow rate is small, and it is easily changed, it is easy to optimizethe quantitative determination by changing the flow rate. As a means oftransportation for this purpose, micropumps may be utilized. Any meansmay be utilized such as electrophoretic flow and electroosmotic flow byapplying en external electric field, as long as it can transport themedium containing analytes to the detecting unit.

By the device for quantitatively determining an analyte according to thepresent invention, it is possible to quantitatively determine an analytein a wide range without lowering the detection sensitivity, greatlyimproving the capacity of quantitative determination of an analyte.Accordingly, it can be effectively used as part of a MEMS or μTAS.

Any known technology may be utilized for manufacturing a device forquantitatively determining an analyte according to the presentinvention. The following are some examples.

First, grooves for the flow channel are formed on a substrate. Anymaterial such as glasses, plastics, semiconducting materials, etc. maybe used for the substrate, as long as it does not contradict the gist ofthe present invention. The grooves for the flow channel may be formed byany technology including mechanical processing and etching technologiesbelonging to the semiconductor processing technologies. It is preferableto cover the surface of the substrate to prevent the solution containinganalytes from evaporating or scattering. Needless to say, the covershould be made of a material that is transparent in the wavelength foruse in the observation, when it is necessary to optically observe theflow channel.

A detecting unit for detecting an analyte to which a capturing unit isattached is installed on a part of the flow channel. Any material may beused for the detecting unit according to the present invention, as longas it does not contradict the gist of the present invention, and thereis no limitation to the shape.

As an analyte according to the present invention, any material may beused as long as it can be quantitatively determined by the device forquantitatively determining an analyte according to the presentinvention. As an analyte, preferable is a material selected from thegroup consisting of proteins, DNAs, RNAs, antibodies, natural orartificial single-stranded nucleotides, natural or artificialdouble-stranded nucleotides, aptamers, products obtained by limiteddecomposition of antibodies with a protease, organic compounds havingaffinity to proteins, biomacromolecules having affinity to proteins,complex materials thereof, ionic polymers charged positively ornegatively, and arbitrary combinations thereof. Examples of theabove-described complex materials according to the present invention mayinclude combined materials from DNAs and negatively-charged polymers,and combined materials from the above-described materials and othermaterials.

Any type of binding can be utilized for the capturing of an analyte aslong as it does not contradict the gist of the present invention,including biological binding, electrostatic binding, physicaladsorption, chemical adsorption, etc., as well as chemical bonding suchas covalent bonding and coordinate bonding.

For example, glasses, ceramics, plastics, metals, etc. can be used for adetecting unit, to install a capturing unit for capturing an analyte onthe surface. The detecting unit may be single-layered or multilayered.It may have a structure other than that of a layer or layers.

Any material can be arbitrarily chosen for the detecting unit dependingon the purpose, but Au is particularly preferable. When it is used foran electrode, it is easy to take out electric signals, and to provideelectric potential to facilitate capturing of analytes. It is also ofteneasy to fix capturing units on a detecting unit.

When the detecting unit can capture an analyte without specificallyforming a capturing unit, it is not necessary to install a capturingunit on the surface of the detecting unit. Taking a case in which ananalyte comprises a nucleotide, and can be bound with a Au layerdirectly via its thiol group for example, there is a device forquantitatively determining an analyte, wherein an analyte 19 having afluorescent signaling unit 61 is bound to a Au electrode (detecting unit13) installed on a sapphire substrate 62 as shown in FIG. 6, by reactingthe nucleotide with the polished Au electrode at room temperature for 24hours. “S” which is located in the lower portion of the single-strandedoligonucleotide structure represents that the analyte 19 is directlybound with the Au electrode 13 via a thiol group.

Any material may be used as the capturing unit, as long as it does notcontradict the gist of the present invention. It is preferable that thematerial has a property to specifically bind to the above-describedanalyte. For example, if the analyte is a DNA, it is preferable that thecapturing unit has a function to specifically bind to the DNA, and ifthe analyte is a protein, it is preferable that the capturing unit has afunction to specifically bind to the protein.

Preferably, such a capturing unit comprises at least one materialselected from the group consisting of proteins, DNAs, RNAs, antibodies,natural or artificial single-stranded nucleotides, natural or artificialdouble-stranded nucleotides, aptamers, products obtained by limiteddecomposition of antibodies with a protease, organic compounds havingaffinity to proteins, biomacromolecules having affinity to proteins,complex materials thereof, ionic polymers charged positively ornegatively, and arbitrary combinations thereof. Examples of theabove-described complex materials in the present invention may includecombined materials from DNAs and negatively-charged polymers, andcombined materials from the above-described materials and othermaterials.

Hereupon, the “nucleotide” according to the present invention is any oneselected from the group consisting of mononucleotide, oligonucleotidesand polynucleotides, or a mixture thereof. Such materials are oftennegatively charged. Single-stranded nucleotides and double-strandednucleotides can be used. They can be specifically bound with analytesthrough hybridization. Proteins, DNAs and nucleotides can be used as amixture. The biomacromolecules include those derived from livingorganisms, those processed from materials derived from living organisms,and synthesized molecules.

Hereupon, the above-described “products” are those obtained by limiteddecomposition of antibodies with a protease, and can comprise anything,as long as they conform to the gist of the present invention, includingFab fragments or (Fab)₂ fragments of antibodies, fragments derived fromFab fragments or (Fab)₂ fragments of antibodies, derivatives thereof,etc.

As an antibody, monoclonal immunoglobulin IgG antibodies can be used,for example. Fab fragments or (Fab)₂ fragments of IgG antibodies can beused as fragments derived from IgG antibodies, for example. Furthermore,fragments derived from those Fab fragments or (Fab)₂ fragments can beused. Examples of applicable organic compounds having affinity toproteins are enzyme substrate analogs such as nicotinamide adeninedinucleotide (NAD), enzyme activity inhibitors, neurotransmissioninhibitors (antagonist), etc. Examples of biomacromolecules havingaffinity to proteins are proteins that can act as a substrate or acatalyst for proteins, element proteins constituting molecularcomposites, etc.

It is sufficient if the total number of capturing units to be attachedto is sufficiently larger than the number of expected analytes. However,from the viewpoint of detection sensitivity, it is preferably from twiceto ten times as large as that of analytes that is expected. When thereare plural kinds of analytes as the objects for the quantitativedetermination, the number of the capturing units should be determined inreference to the number of each kind of analyte.

A device for quantitatively determining an analyte according to thepresent invention manufactured according to the way of this example, issuitable for determining a tiny amount of minute analytes, and issuitably utilized for the above-described MEMS and μTAS, for example.Regarding the application, it is suitable for biochips, DNA chips, etc,for example.

A method for quantitatively determining an analyte according to thepresent invention comprises: using a device for quantitativelydetermining an analyte having the above-described functions, or,regarding a different device, using a flow channel, a detecting unit forcapturing and detecting the analyte, and a quantitative measurement unitfor quantitatively determining the analyte; and dividing a signalgenerated when the detecting unit has detected the analyte, into aplurality of parts in the direction of the flow in the flow channel atthe quantitative measurement unit for processing. By this method, thequantitative determination of an analyte can be performed in a widerange without lowering the detection sensitivity, and the performance inquantitative determination of an analyte can be conspicuously improved,in the same way as has been explained about the device forquantitatively determining an analyte according to the presentinvention.

In this case, variations such as using a plurality of devices in seriesor in parallel, and installing accessories, may be applied. In such acase, there would be an increasing number of defects such as complicatedequipment and more time consumed for the measurement, compared with acase in which a device for quantitatively determining an analyteaccording to the present invention, is used. Nevertheless, it may bepossible to accomplish effects similar to those achieved when the devicefor quantitatively determining an analyte according to the presentinvention, is used.

Regarding this embodiment of the present invention, preferable are thatthe detecting unit has a capturing unit for capturing the analyte; thata plurality of detecting units are disposed in the direction of the flowin the flow channel; that the quantitative measurement unitquantitatively determines the analyte, using an optical signal orelectric signal; and that a plurality of capturing units forspecifically capturing different analytes, are disposed on the detectingunit.

In the quantitative determination, it is possible to improve theperformance in the quantitative determination by optimizing the numberof analytes captured by the detecting unit, in the direction of the flowin the flow channel, as has been already explained regarding the devicefor quantitatively determining an analyte according to the presentinvention. Specifically, it is possible to do the optimization, bychanging at least one factor selected from the group consisting of thesupply rate of analytes, the length of a detecting unit in the directionof the flow, the number of detecting units in the direction along thewidth of the flow channel, and the thickness of the flow channel.

Furthermore, it is preferable that the detecting unit is an electrode,and an electric potential for electrically attracting a charged analyteto the electrode is applied to the electrode, in order to rapidlycapture the analyte by the capturing unit. In such a case, the electricpotential may be determined appropriately, in consideration of the timerequired for the quantitative determination and the precision of thequantitative determination.

It is useful to change the electric potential for electricallyattracting a charged analyte to the electrode according to the locationof the electrode in the direction of the flow. As the concentration ofanalyte in the medium is lowered as the flow goes downstream, thecapturing efficiency of analyte is lowered. Accordingly, it is useful tochange the electric potential according to the location of theelectrode, and increase the attracting effect of the electric potentialas the flow goes downstream, so that the sensitivity is prevented frombeing lowered.

It is to be noted that the method for quantitatively determining ananalyte according to the present invention is particularly useful whenthe analyte is a DNA, and the capturing unit has a function to bespecifically bound to a DNA, and when the analyte is a protein, and thecapturing unit has a function to be specifically bound to a protein.

Next, the explanation on the second aspect of the present invention willbe made. When required molecules are attached to the surface of a solid,it is a common method to immerse the solid into a solution containingthe molecules. At this moment, electrostatic repulsion between moleculeswill come about, when molecules that are charged (acquiring electriccharge) in a solution, or those that are bound with materials havingelectric charge are used as the attached molecules. Because of this,other molecules are prevented from coming close to the moleculesattached to the surface of a solid by the electrostatic repulsion.

It is to be noted here, that molecules that are charged, molecules thatare bound with materials having an electric charge, or the like aregenerally called simply “molecules that are charged” or “chargedmolecules” in the second aspect of the present invention. The detailwill be explained later.

When an aqueous solution containing an electrolyte is used as thesolvent, the molecules are surrounded with ions (counter ions) having anelectric charge that is opposite to that of the molecules with a certaindistance therebetween that is determined by the probability ofoccurrence, and accordingly, the electric charges are neutralized by theions. This effect of compensating the charges between molecules iscalled the screening effect, and the layer of these counter ions iscalled the diffused electric double layer.

The thickness of the diffused electric double layer is dependent on thesalt concentration of the electrolyte (ion concentration of theelectrolyte), and the higher the salt concentration is, the thinner thelayer is. The thickness of the layer is widely known as the Debye length(L_(Debye)). It can be expressed by the following equation in the caseof a 1:1 type electrolyte (an electrolyte composed of a pair of amonovalent cation and a monovalent anion).L _(Debye)=(2,000N _(A) cz ² e ²/(∈_(r)∈₀ kT)^(−1/2)(m)  (1)

Hereupon, N_(A) is the Avogadro's number, c is the ion concentration ofthe electrolyte (mol/L), z is the valence of the ion, e is a unitcharge, ∈_(r) is the relative dielectric constant of a medium, ∈₀ is thedielectric constant of vacuum, k is the Boltzmann constant, and T is theabsolute temperature.

From this equation, it is possible to easily estimate the change ofL_(Debye) when the ion concentration is changed. FIG. 7 shows the changeof L_(Debye) when the ion concentration of a 1:1 type electrolyte suchas NaCl is changed. It is understood that L_(Debye) is small in a regionwhere the ion concentration is high, and accordingly the screeningeffect is large. That is, it is possible to conclude that theelectrostatic repulsion between charged molecules can be restrained, andaccordingly, a film of charged molecules with a high attaching densitycan be manufactured, in such a region of ion concentration. Furthermore,more precise control of the attaching density is possible, by takinginto consideration, the screening effect and the size of chargedmolecules in the solution.

In this way, it is possible to uniquely calculate L_(Debye), when whatelectrolyte ion is used, is determined. Furthermore, if the size of acharged molecule in a solution is known, it is possible to estimate, bythe calculation, the attaching density of charged molecules that changesaccording to the ion concentration, taking into consideration the sizeand L_(Debye).

An example in which the charged spherical molecules that are negativelycharged and have a radius of 1 nm, are attached to the surface of asolid, will be explained as follows.

When a case is provided where there occurs no electrostatic repulsionbetween charged molecules, and a monomolecular film is adsorbed to aflat surface of a solid, it is possible to consider that a film ofhexagonally arranged molecules is formed as a closest packing structureas shown in FIG. 8. In this case, the attaching density (thickestsurface density) of molecules can be represented, using the radius r ofthe molecule, by:Attaching density=1/(2r ²√{square root over (3)})(cm⁻²)  (2).When the r is 1 nm for the molecule, a constant attaching density of2.9×10¹³ cm⁻² is obtained.

When the static repulsion between charged molecules and L_(Debye) aretaken into consideration, the layer of adsorbed molecules has moleculeshaving a separation of L_(Debye)×2 therebetween as shown in FIG. 9, andthe attaching density can be represented by the following equation.Attaching density=1/(2(r+L _(Debye))²√{square root over (3)})(cm⁻²)  (3)Accordingly, for a case where a 1:1 type electrolyte such as NaCl isused, and the molecule has a radius of 1 nm, it is possible to controlthe attaching density by changing the ion concentration of theelectrolyte, as shown in FIG. 10.

The above is about cases when the size of a charged molecule in asolution is known. However, even if the size is unknown, it is possibleto estimate the size of the unknown molecule from the experimental dataobtained by measuring the attaching density of charged molecules at aspecific ion concentration of an electrolyte through experimentation,and accordingly, to estimate the dependency of the attaching density onthe ion concentration, taking into consideration, the size and screeningeffect.

In other words, in the effective size decision method of chargedmolecules according to the present invention, the effective size of acharged molecule in a solution containing an electrolyte and the chargedmolecules is estimated by the screening effect by the electrolyte.

Specifically for example, the effective size of a charged molecule canbe determined as a radius of a molecule, when the Debye length isdetermined from the ion concentration of the electrolyte, using equation(1), and the Debye length and the measured attaching density data ofcharged molecules are applied to equation (3). The attaching density inthis case can be obtained by a quantitatively determining method usingXPS observing a specific element of the attached molecules, aquantitatively determining method wherein radioactive labels areattached to the attached molecules beforehand, a quantitativelydetermining method to measure the redox current of a redox marker toneutralize the charge of the charged, attached molecules, etc.

Any material may used as the electrolyte for use in this case, as longas it does not contradict the gist of the present invention. It ispreferably a 1:1 type electrolyte such as NaCl or KCl, from theviewpoint of simplifying the effect on the charged molecules. A mixturethereof may be used.

The “charged molecule” according to the second aspect of the presentinvention includes molecules that simply have a charge as explainedearlier, those having acquired a charge as a cluster of molecules havingan electric charge or not having an electric charge, and a materialhaving an electric charge, as a result of the molecules being bound withthe material, and those acquired a charge as a cluster of moleculeshaving an electric charge and a material not having an electric charge,as a result of binding of the molecules with the material. In the lattertwo cases, a cluster is the “charged molecule” according to the presentinvention. In this case, the “binding” may be chemical binding orphysical binding. The former is better because the binding stability ishigher and is preferable.

The above-described material is preferably selected from the groupconsisting of proteins, DNAs, RNAs, antibodies, natural or artificialsingle-stranded nucleotides, natural or artificial double-strandednucleotides, aptamers, products obtained by limited decomposition ofantibodies with a protease, organic compounds having affinity toproteins, biomacromolecules having affinity to proteins, complexmaterials thereof, ionic polymers charged positively or negatively, andarbitrary combinations thereof. Molecules that can be bound to such amaterial preferably have a property to be specifically bound to thematerial.

Therefore, the “charged molecule” according to the present inventionpreferably comprises a material selected from the group consisting ofproteins, DNAs, RNAs, antibodies, natural or artificial single-strandednucleotides, natural or artificial double-stranded nucleotides,aptamers, products obtained by limited decomposition of antibodies witha protease, organic compounds having affinity to proteins,biomacromolecules having affinity to proteins, complex materialsthereof, ionic polymers charged positively or negatively, and arbitrarycombinations thereof. The “charged molecule” according to the presentinvention preferably comprises a thiol group to facilitate attachment toa substrate.

Hereupon, the “nucleotide” according to the present invention is any oneselected from the group consisting of mononucleotide, oligonucleotidesand polynucleotides, or a mixture thereof. Such materials are oftennegatively charged. Single-stranded nucleotides and double-strandednucleotides can be used. They may be hybridized to be part of chargedmolecules. Proteins, DNAs and nucleotides can be used as a mixture. Thebiomacromolecules include those derived from living organisms, thoseprocessed from materials derived from living organisms, and synthesizedmolecules.

Hereupon, the above-described “products”, the “antibody”, organiccompounds having affinity to proteins, and biomacromolecules havingaffinity to proteins, have each the same meaning and may have the sameexamples as described before.

It is to be noted that the “charged molecule” according to the presentinvention may acquire a role to make the surface of a substrate eitherhydrophobic or hydrophilic, by the attaching to the substrate, and arole to provide functions which the original surface of a solid has nothad, by the adsorption of special molecules such as DNAs and proteins tothe surface. A specific example is formation of a monolayer film havinga functionality, by making the surface hydrophobic by covering it with—CH₃, making the surface hydrophilic by covering it with NH³⁺ or COO⁻ toform either a positively charged surface or a negatively chargedsurface, making the surface adsorb DNAs or proteins, or by doing asimilar modification.

In the method for attaching charged molecules onto a substrate accordingto the present invention, an electrolyte is made present in the solutioncontaining the molecules, and the screening effect by the electrolyte isadjusted in order to attach charged molecules to the substrate. Theeffective size of a molecule in the solution is also taken intoconsideration.

For example, because the attaching density, the Debye length andeffective size of a molecule are interconnected with each other throughequation (3), it is possible to calculate a desired Debye length fromthe effective size of a molecule to be attached and a desired attachingdensity, and determine the kind of an electrolyte and its ionconcentration to meet the Debye length. Accordingly, it is possible tomake molecules attach to a substrate so that the surface of thesubstrate has the required attaching density.

In this case, if the effective size of a molecule is known, the size maybe used. When the effective size of a molecule is unknown, the effectivesize of a charged molecule in a solution containing an electrolyte andthe charged molecules may be estimated from the screening effect by anelectrolyte, as described above. As an electrolyte in this case, a 1:1type electrolyte is preferably used.

When this method is employed, it is possible to obtain a device toattach charged molecules to a substrate in which the density ofmolecules attached to the substrate can be controlled. Any device may beused for the device as long as it meets the object of the presentinvention. For example, this device may be installed as a part oraccessory of a MEMS that is a precision system in which a plurality offunctional parts such as mechanical, optical and hydrodynamic parts areintegrated and miniaturized, or a μ-TAS that is a chemical analysissystem with micropumps, microvalves, sensors or the like, miniaturized,accumulated, and integrated, both systems being manufactured, usingtechnologies for preparing very small devices, based on semiconductorprocessing technologies. As a MEMS or μ-TAS, specifically enumerated arean ion sensor such as a pH sensor and gas sensor, and a device fordetecting molecules such as a DNA chip and protein chip for use indetecting wholly or partly charged molecules such as DNAs and proteins.It is to be noted that “detection” in the present invention includesdetermining the presence or absence of charged molecules or moleculesthat are specifically bound to a material that is bound with the chargedmolecules, and determining the kind, size and/or amount of chargedmolecules or the material that is bound with the charged molecules.

Using this device, it is possible to observe the motion of chargedmolecules in a state in which there is no interactive influence betweenthe charged molecules or to evaluate the interactive influence betweencharged molecules, by selecting a desired value of the attaching densityof charged molecules on a substrate.

As a molecule detecting device for use in detecting charged molecules soas to observe the motion of the charged molecules in a state in whichthere is no interactive influence between the charged molecules, it ispreferable to use the above-described method for attaching molecules toa substrate, and use, as the molecule detecting unit, a substratewhereby the attaching density is controlled so that the distance betweenadjacent molecules attached to the substrate is not less than twice theeffective length of a molecule, preventing the molecules from physicallycontacting each other.

In addition, as a molecule detecting device for use in detecting chargedmolecules in order to evaluate the interactive influence between chargedmolecules, it is preferable to use the above-described method forattaching molecules to a substrate, and use, as the molecule detectingunit, a substrate whereby the attaching density is controlled so thatthe distance between adjacent molecules attached to the substrate isless than twice the effective length of a molecule, making the moleculesphysically contact each other.

It is to be noted that the above distance between adjacent molecules canbe determined from the attaching density.

In such a device for detecting molecules, any known method may be usedas a method for detecting molecules. Examples are a method in whichvoltage is applied between a substrate and a solution containing anelectrolyte, and an electric signal is taken out as the response, amethod in which a fluorescent pigment is attached to charged molecules,and the fluorescence is detected, or similar other methods.

FIG. 13 shows an example of a device for detecting molecules, wherein afluorescent pigment is attached to charged molecules for thefluorescence to be detected. The device 1 for detecting molecules inFIG. 13 shows states of a charged molecule 5 having a fluorescentpigment 4 in an extended state (on the left), and a charged molecule 5having a fluorescent pigment 4 in a contracted state (on the right), ona Au electrode 3 (corresponding to a substrate according to the presentinvention) installed on a base 2. The charged molecule 5 in thecontracted state can be changed into one in an extended state, byapplying a certain potential difference between the Au electrode 3 and acounter electrode 8 with an external electric field applying device 9.By this, the distance between the fluorescent pigment 4 and the Auelectrode 3 changes. At this moment, if light 11 is irradiated from alight irradiating device 10, fluorescence 12 is obtained from thecharged molecule 5 in the extended state (on the left).

Using the device for detecting molecules according to the presentinvention, it is possible to control the attaching density of moleculesattached to the surface of a solid, and accordingly, it is possible toclarify the structure and/or behavior of molecules attached to thesurface of a solid in a solution, and to realize a device usingmolecules attached to the surface of a solid with high reliability andhigh sensitivity.

For example, if a DNA or antibody is used for charged molecules attachedto the surface of a substrate, and the density is quantitativelycontrolled, the structure and/or behavior of the targeted DNA or proteinin an aqueous solution can be clarified. Also, by optimizing thedetection conditions through these pieces of information, devices withhigh reliability and high sensitivity can be realized. Accordingly, itwill be easy to evaluate the presence or absence of a target, and/or todetermine the kind, size and/or amount of the target.

In addition, the technology of the present invention is very effectivein the field of biosensors such as MEMSs or μTASs, since it is necessaryto provide a state in which these molecules are sufficiently apart fromeach other (that is, a state in which there is no steric hindrance),with a high reproducibility, when the observation of the electric and/orphysical properties of each molecule on a functional surface is appliedto those biosensors.

Furthermore, when the interactive action between a molecule and thesurrounding molecules is necessary in such cases as bonding betweenattached molecules, exchanging of charges between molecules, opticalenergy transfer, etc., it is necessary for these molecules to besufficiently close to each other, in contrast with the above. Thetechnology of the present invention is also very effective in such afield.

Furthermore, the technology of the present invention can greatlycontribute to the elucidation of functions of charged molecules withunknown behaviors in an aqueous solution. Accordingly, discovery of newfunctional materials and functional surfaces may be highly expected.

It is to be noted that for the substrate according to the presentinvention, any of electroconductive materials such as metals,semiconducting materials such as silicon, and insulating materials suchas glasses, ceramics, plastics and sapphire, may be used, according topurposes. As the electroconductive material, an electroconductivesubstance itself as well as one having an electroconductive layer on thesurface of a glass, ceramic, plastic or metal may be used. As theelectroconductive substance, any substance may be used, including anelementary metal, alloy, or laminate. Noble metals represented by Au ispreferably used as they are chemically stable.

EXAMPLES

The present invention will be explained in detail in reference to thefollowing examples.

Example 1

This example relates to a DNA chip wherein a single-stranded DNA isinstalled on a detecting unit as a capturing unit, to detect theconcentration of a complementary DNA or an analyte in an aqueoussolution.

The outline of an exemplary DNA chip of this example and its basicprinciple of operation will be explained, using FIGS. 4-A to 4-C. First,as shown in FIG. 4-A, a Au thin film is installed as a detecting unit 13at the bottom of part of a 1-mm wide flow channel in an elongated shapealong the flow channel. A single-stranded DNA 42 having an SH group onone end as a capturing unit is attached onto the Au thin film 13 at asurface density of 5×10¹² molecules/cm² (see Analytical Chemistry, vol.70, p. 4670-4677, 1998, for example), to make capturing units. Atwo-dimensional CCD light-receiving unit 41 is installed over the flowchannel with the single-stranded DNA attached thereto, for observingfluorescent signals from the capturing units.

Next, as shown in FIG. 4-B, an aqueous solution containing acomplementary DNA 43 as an analyte with a fluorescent label 44 is madeto flow into the flow channel, to hybridize with the single-stranded DNA42 to form double strands as shown in FIG. 4-C. Regarding the units thathave formed double strands, fluorescence from the fluorescence labels 44attached to the complementary strands is detected by the light-receivingunit 41.

As the aqueous solution containing an analyte flows into the detectingunit from the upstream side, the formation of the double strands startsat the upstream side of the detecting unit, and advances towards thedownstream side until the analyte is consumed. Because the number ofcapturing units on the detecting unit is determined through theabove-described surface density, the number of the complementary DNA oranalyte, that is, the concentration can be determined from the length ofthe capturing units detected through the fluorescent signals.

In this example, the width of the flow channel is 1 mm. Therefore, whenthe fluorescent signals are observed 5 mm long along the direction ofthe flow in the flow channel, the area in which the fluorescent signalsare observed is 0.05 cm², and accordingly, the absolute number of thecomplementary DNA molecules is 5×10¹² molecules/cm²×0.05 cm²=2.5×10¹¹molecules. Accordingly, if the volume of the used solution containingthe complementary DNAs is, for example, 100 μL, the originalconcentration of the analyte or the complementary DNA, can be determinedas 2.5×10¹¹ molecules/(0.1 cm³)=2.5×10¹²/cm³ molecules.

Example 2

This example relates to determining the effective radius of asingle-stranded oligonucleotide in an aqueous solution, and further, tocontrolling the attaching density of oligonucleotide attached to asubstrate.

Synthesized was a single-stranded 24-mer oligonucleotide to which athiol group having a C₆H₁₂ alkyl chain (—C₆H₁₂—SH) at the 3′ terminalwas introduced, and the thiol group was reacted with a polished Auelectrode at room temperature for 30 minutes, to bind thesingle-stranded oligonucleotide to the electrode. The thiol group may beintroduced to an intermediate section of a single strand or at the 5′terminal. The aqueous solution used for the reaction contained 10 mM ofa Tris buffer (2-amino-2-hydroxymethylpropane-1,3-diol), and adjusted tohave a pH of 7.4.

NaCl was used as an electrolyte. The screening effect of a negativelycharged oligonucleotide was controlled, by changing the ionconcentration of the electrolyte (salt concentration). Since theeffective size of the oligonucleotide in the aqueous solution wasunknown, while the structure was known, the attaching density of theoligonucleotide attached to the surface of the Au electrode wasmeasured, by reacting the thiol group of the single-strandedoligonucleotide with the Au electrode beforehand, under the saltconcentration conditions of 3 mM, 500 mM, and 1,000 mM.

FIG. 11 is a graph showing the relationship between the attachingdensity of single-stranded oligonucleotide molecules and the ionconcentration of the electrolyte, and the relationship between the Debyelength and the ion concentration of the electrolyte. The ◯ (outlinedcircle) marks in FIG. 11 are attaching density data determined throughexperimentation. As a result of calculation by fitting the experimentalresults onto the calculated curves obtained from equations (1) and (3),the effective size of the 24-mer oligonucleotide in the aqueous solutionwas decided to be 1.9 nm in radius.

To obtain an electrode surface that is controlled to have a desiredattaching density of 3×10¹² molecules/cm² from this calculated curve, itis understood that the salt concentration must be controlled to be 50mM, based on the curve obtained by the calculation. The ● (solid circle)mark in the figure is an experimental data when the oligonucleotide wasattached at the salt concentration of 50 mM. From the calculation, it ispossible to easily determine the salt concentration condition to obtaina desired attaching density.

In addition, ▪ (solid square) marks show the result of attachingexperimentation when a single-stranded, 12-mer oligonucleotide having asimilar thiol group was used. As a result of calculation by fitting theexperimental results onto the calculated curves obtained from equations(1) and (3), it was found that the molecule also had an effective sizesimilar to that of the 24-mer in an aqueous solution. Thesingle-stranded, 12-mer oligonucleotide and the single-stranded, 24-meroligonucleotide are each molecule in the shape of a strand. Accordingly,from the fact that the Debye length's are not different from each other,it is considered that in a case where a strand-type molecule one end ofwhich is modified with a thiol group is chemically adsorbed onto asubstrate, the length of the molecule does not influence the effectivesize and Debye length.

In this way, by utilizing the present invention, it is possible not onlyto control the attaching density of charged molecules such asoligonucleotide molecules, but also to estimate the effective size of anunknown molecule in aqueous solutions, and to collect information on thestate of the attaching.

Example 3

As a related example, the above-described method was employed, andsingle-stranded, 12-mer oligonucleotide molecules having a fluorescentpigment at an end, and a thiol group at the other end were attached to aAu electrode. The result of monitoring the fluorescence from the pigmentwith varying the density of the attached molecules is shown in FIG. 12.This fluorescent pigment decreases or extinguishes the light by thequenching effect when it comes close to or contacts with the Ausubstrate, and emits or increases the light when it goes away from thesubstrate.

From FIG. 12, it is evident that by varying the attaching density, thereappear three regions observed: (1) a low density region where adjacentoligonucleotide molecules do not interfere with each other, and thefluorescence intensity is dependent only on the increase in theattaching density; (2) a intermediate density region where sterichindrance between adjacent oligonucleotide molecules occurs, forcing theoligonucleotide molecules to uprise, with the result that the quenchingeffect on the Au substrate is made smaller, and therefore, thefluorescence intensity is greatly increased owing to the increase in theattaching density and the decrease in the quenching effect; and (3) ahigh density region where almost all oligonucleotide molecules areuprising, the quenching effect does not change, and the fluorescenceintensity is again dependent only on the increase in the attachingdensity.

From these result, it is possible, by utilizing the present invention,to provide a device in which the attaching density is controlled inregion (1), when the device is used for applications where a free motionof oligonucleotide molecules is permitted, without being hindered bysurrounding nucleotide molecules. It is also possible to provide adevice in which the attaching density is controlled in region (2) or(3), when the interactive actions with surrounding molecules arenecessary in such a case as one in which attached molecules are boundtogether, one in which electric charges are exchanged or opticalenergies are transferred between molecules, or the like.

What is claimed is:
 1. A method for attaching molecules having anelectric charge to a substrate, comprising: attaching said molecules tosaid substrate by bringing a solution containing said molecules intocontact with said substrate; having an electrolyte having an ionconcentration also present in the solution; and selecting a density ofsaid molecules on said substrate by adjusting the ion concentration ofthe electrolyte to influence an effective size of said molecule attachedto the substrate when said molecules having an electric charge areattached to the substrate, wherein: said effective size attached to thesubstrate is a sum of the radius of said molecule and Debye lengthaccording to the following equation,L _(Debye)=(2,000N _(A) cz ² e ²/(∈_(r)∈₀ kT))^(−1/2)(m), and saiddensity is determined by the following equation,Density=1/(2(r+L _(Debye))²√3)(cm⁻²) where L_(Debye) is the Debyelength, N_(A) is Avogadro's number, c is the ion concentration of theelectrolyte (mol/L), z is the valence of the ion, e is a unit charge,∈_(r) is the relative dielectric constant of a medium, ∈₀ is thedielectric constant of vacuum, k is the Boltzmann constant, T is theabsolute temperature, and r is the radius of the molecule, wherein saidmolecule having an electric charge comprises a strand-type materialselected from the group consisting of proteins, DNAs, RNAs, antibodies,natural or artificial single-stranded nucleotides, natural or artificialdouble-stranded nucleotides, aptamers, products obtained by limiteddecomposition of antibodies with a protease, and arbitrary combinationsthereof.
 2. The method of claim 1, wherein said electrolyte comprises amonovalent cation and a monovalent anion.
 3. The method of claim 2,wherein said electrolyte comprising a monovalent cation and a monovalentanion is NaCl, KCl or a mixture thereof.